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Method for detecting Parp activity based on analysis of hemin-graphene composites

A composite material, graphene technology, applied in the measurement of color/spectral properties, etc., can solve problems such as cumbersome synthesis, and achieve the effects of simplified detection methods, low cost, and sensitivity and specificity

Active Publication Date: 2020-04-03
HENAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires tedious synthetic
Therefore, this method has many limitations

Method used

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  • Method for detecting Parp activity based on analysis of hemin-graphene composites
  • Method for detecting Parp activity based on analysis of hemin-graphene composites
  • Method for detecting Parp activity based on analysis of hemin-graphene composites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Based on the analytical method for analyzing and detecting PARP activity based on H-GNs composite material, the detection steps are:

[0042] Synthesis steps of H-GNs composite material: Weigh 10 mg of graphite oxide and dissolve it in 20 mL of secondary water (0.5 mg / mL), ultrasonically disperse for 2 h for mechanical exfoliation, centrifuge at 3000 rpm for 30 min to remove unexfoliated graphite oxide, and take the supernatant in dialysis The bag (MW:8000-12000) was dialyzed for one week to remove impurities and small molecules to obtain uniformly dispersed graphene oxide. The resulting graphene oxide solution was thoroughly mixed with 20 mL of hemin (0.5 mg / mL) dissolved in 0.1 M NaOH solution in a flask. After completion, slowly add 200 μL of ammonia solution and finally 30 μL of hydrazine hydrate. The mixed solution was vigorously stirred for 60 minutes, and the flask was placed in a water bath (60° C.) for 20 hours to obtain a stable and dispersed black solution. ...

Embodiment 2

[0053] The difference from Example 1 is: select different concentrations of PARP (U, 1U=45ng): (a) 0 (b) 0.05 (c) 0.1 (d) 0.3 (e) 0.5 (f) 0.75 (g) 1 ( h)2(i)3, the obtained UV-visible light curve is as follows Figure 5 As shown, it can be seen that PARP has a good linear relationship between 0.05U and 1U in the range of 0-3U.

[0054] In summary, H-GNs composites can quantitatively detect PARP, due to the intrinsic catalase-like activity of H-GNs composites, adjustable dispersion in saline solution, and effective use of the characteristics of nanomaterials, The detection method does not need to be detected by precision instruments, the detection method is simplified, and the cost of virus detection is greatly reduced. The invention can successfully detect the activity of PARP added in serum, and has good clinical significance.

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Abstract

The invention discloses a method for analyzing and detecting PARP (poly-ADP-ribose polymerase) activity based on a hemin-graphene composite material. The method comprises the following steps: (1) selecting activated DNA; (2) synthesizing an H-GNs (hemin-graphene) composite material; (3) mixing the activated DNA, PARP and nicotinamide adenine dinucleotide (NAD<+>) for reaction, thus catalyzing the PARP to synthesize a PAR (poly ADP-ribose) polymer with a large number of negative charges; (4) enabling H-GNs and the product PAR polymer to react, adding a saline solution, and recording an agglomeration change of the H-GNs; (5) detecting a product solution by using an ultraviolet-visible spectrograph. By using electrostatic interaction between the H-GNs and the product PAR polymer, colorimetric reaction caused by the agglomeration change of the H-GNs in the saline solution is generated, and the activity of the PARP can be detected quantitatively. The method has the advantages of simpleness and convenience, quick speed, high sensitivity and no need of DNA probe marking.

Description

technical field [0001] The invention belongs to a technique for quantitatively detecting PARP (polyadenylate diphosphate-ribose polymerase) activity, relates to the application of graphene composite materials in clinical detection, and specifically relates to the application field of graphene composite materials for quantitative detection of biological enzymes. Background technique [0002] PARP, also known as poly ADP-ribose polymerase, is a class of nuclear enzymes that can catalyze poly ADP ribosylation in most eukaryotes. Its structure mainly includes three regions: DNA binding domain, self-modification domain, catalytic domain. PARP is able to bind various DNA structures and nucleosomes, and has NAD + Depending on the catalytic activity, poly(ADP-ribose) polymers can be synthesized on target proteins. The formation of poly(ADP-ribose) polymers significantly alters the charge properties of the receptor protein, triggering DNA damage control and repair processes. PARP ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 刘勇徐晓林卫伟
Owner HENAN UNIVERSITY
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